Cells that secrete molecules are an important source of therapeutically important biological molecules, and thus it is often valuable to screen secreted molecules from a population of secreting cells for certain functional properties. Useful secreting cells may be, for example, murine or human plasma cells or other antibody secreting cells, where the secreted antibody may block the binding of a cytokine to its cognate receptor thereby disrupting a signal transduction event that may underlie the physiopathology of a disease. Therefore, such blocking antibodies possess tremendous therapeutic value and hence are of interest to the pharmaceutical industry. Alternatively, engineered bioactive molecules such as a cytokine with a variant amino acid at a strategic residue can be screened for enhanced binding to a cognate receptor. Currently, such functional screening is a laborious procedure typically done with a large number of cells in 96-well plates.
Much work has been done to engineer cells to “report” when a ligand binds to a receptor expressed on the surface of the cells. Engineering has been desirable because the cells may not have readily assayable changes in response to such a binding event. Therefore, certain genes are chosen as reporters because the characteristics they confer when expressed are easily identified and measured. Some reporter cells are comprised of reporter genes encoding readily assayable proteins under the control of well-characterized transcriptional regulatory elements responding as part of a well-known signal transduction pathway. Examples of common reporter genes are LacZ (encoding beta-galactosidase), luc (encoding firefly luciferase), luxCDABE (encoding bacterial luciferase) and GFP (encoding jellyfish green fluorescent protein) (Ghim et al., BMB reports, 2010, 451:460). One of the most commonly used signal transduction pathways is the NF-κB activation pathway. Reporter genes are typically placed under the control of NF-κB regulatory elements and are used to screen molecules interacting with the relevant receptor, which causes activation of NF-κB and is manifested by expression of the reporter genes.
There are many limitations to each of the aforementioned reporter genes. The lacZ assay requires expensive and potentially toxic chemicals, the luc assay requires expensive luciferin, bacterial luciferase cannot be used in eukaryotic cells, and GFP is so stable it cannot be used to report short term negative processes where the GFP signal needs to diminish quickly (Ghim et al., supra). Since the above-stated reporter systems typically are used on a large population of cells, it is difficult to employ them in nanofabricated constructs on a limited number of cells. Furthermore, these reporter assays require translation of the protein products to visualize the output, which often takes hours or even up to a day.
In addition to the above-mentioned reporter systems utilizing expressed proteins, microarrays have been used to investigate multi-gene mRNA expression between multiple populations of tissues or cells. Microarrays allow many mRNA sequences to be sampled at the same time, but because of the expense of preparation and handling the number of tissue samples is generally small (less than 20) and the cell population large (more than a million cells). Therefore, the use of mRNA expression as a reporter system is a serial and labor intensive process.
In order to correct the deficiencies of the current state of the art, a method is needed where a single cell secreting a biologically active moiety may be tested by one or more reporter cells and/or one or more types of reporter cells, and where an artificial reporter gene(s) does not need to be engineered into the reporter cell(s) to read out the response upon a binding event between a receptor and the active moiety, or upon disruption of binding. The present invention generally relates to methods, devices and kits directed to these and similar utilities, such methods, devices and kits being further described herein.